The present disclosure relates to isolated polynucleotides encoding a delta-9 elongase, delta-9 elongases encoded by the isolated polynucleotides, expression vectors comprising the isolated polynucleotides, host cells comprising the expression vectors, and methods for producing delta-9 elongases and polyunsaturated fatty acids.
Polyunsaturated fatty acids (PUFAs) play many roles in the proper functioning of life forms. For example, PUFAs are important components of the plasma membrane of a cell, where they are found in the form of phospholipids. They also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins. Additionally, PUFAs are necessary for the proper development of the developing infant brain as well as for tissue formation and repair. In view of the biological significance of PUFAs, attempts are being made to efficiently produce them, as well as intermediates leading to their production.
A number of enzymes, most notably desaturases and elongases, are involved in PUFA biosynthesis (see FIG. 1). Desaturases catalyze the introduction of unsaturations (e.g., double bonds) between carbon atoms within the fatty acid alkyl chain of the substrate. Elongases catalyze the addition of a 2-carbon unit to a fatty acid substrate. For example, linoleic acid (LA, 18:2n-6) is produced from oleic acid (OA, 18:1n-9) by a Δ12-desaturase. Eicosadienoic acid (EDA, 20:2n-6) is produced from linoleic acid (LA, 18:2n-6) by a Δ9-elongase. Dihomo-γ-linolenic acid (DGLA, 20:3n-6) is produced from eicosadienoic acid (EDA, 20:2n-6) by a Δ8-desaturase. Arachidonic acid (ARA, 20:4n-6) is produced from dihomo-γ-linolenic acid (DGLA, 20:3n-6) by a Δ5-desaturase (see FIG. 1).
Elongases catalyze the conversion of γ-linolenic acid (GLA, 18:3n-6) to dihomo-γ-linolenic acid (DGLA, 20:3n-6) and the conversion of stearidonic acid (SDA, 18:4n-3) to eicosatetraenoic acid (ETA, 20:4n-3). Elongase also catalyzes the conversion of arachidonic acid (ARA, 20:4n-6) to adrenic acid (ADA, 22:4n-6) and the conversion of eicosapentaenoic acid (EPA, 20:5n-3) to ω3-docosapentaenoic acid (22:5n-3). Δ9-elongase elongates polyunsaturated fatty acids containing unsaturation at the carbon 9 position. For example, Δ9-elongase catalyzes the conversion of linoleic acid (LA, 18:2n-6) to eicosadienoic acid (EDA, 20:2n-6), and the conversion of α-linolenic acid (ALA, 18:3n-3) to eicosatrienoic acid (ETrA, 20:3n-3). ω3-ETrA may then be converted to ω3-ETA by a Δ8-desaturase. ω3-ETA may then be utilized in the production of other polyunsaturated fatty acids, such as ω3-EPA, which may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.
The elongases which have been identified in the past differ in terms of the substrates upon which they act. Furthermore, they are present in both animals and plants. Those found in mammals have the ability to act on saturated, monounsaturated and polyunsaturated fatty acids. In contrast, those found in plants are specific for saturated or monounsaturated fatty acids. Thus, in order to generate polyunsaturated fatty acids in plants, there is a need for a PUFA-specific elongase.
In both plants and animals, the elongation process is believed to be the result of a four-step mechanism (Lassner et al., The Plant Cell 8:281-292 (1996)). CoA is the acyl carrier. Step one involves condensation of malonyl-CoA with a long-chain acyl-CoA to yield carbon dioxide and a β-ketoacyl-CoA in which the acyl moiety has been elongated by two carbon atoms. Subsequent reactions include reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA, and a second reduction to yield the elongated acyl-CoA. The initial condensation reaction is not only the substrate-specific step but also the rate-limiting step.
It should be noted that animals cannot desaturate beyond the 49 position, and therefore cannot convert oleic acid (OA, 18:1n-9) into linoleic acid (LA, 18:211-6). Likewise, α-linolenic acid (ALA, 18:311-3) cannot be synthesized by mammals, since they lack Δ15-desaturase activity. However, α-linolenic acid can be converted to stearidonic acid (SDA, 18:4n-3) by a Δ6-desaturase (see WO 96/13591; see also U.S. Pat. No. 5,552,306), followed by elongation to eicosatetraenoic acid (ETA, 20:4n-3) in mammals and algae. This polyunsaturated fatty acid (i.e., ETA, 20:4n-3) can then be converted to eicosapentaenoic acid (EPA, 20:5-3) by a Δ5-desaturase. Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbons 12 (see WO 94/11516 and U.S. Pat. No. 5,443,974) and 15 (see WO 93/11245). The major polyunsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or α-linolenic acid. In view of the inability of mammals to produce these essential long-chain fatty acids, it is of significant interest to isolate genes involved in PUFA biosynthesis from species that naturally produce these fatty acids and to express these genes in a microbial, plant or animal system which can be altered to provide production of commercial quantities of one or more PUFAs. Consequently, there is a definite need for elongase enzymes, the genes encoding the enzymes, as well as recombinant methods of producing the enzymes.
In view of the above discussion, a definite need also exists for oils containing levels of PUFAs beyond those naturally present as well as those enriched in novel PUFAs. Such oils can be made by isolation and expression of elongase genes.
One of the most important long-chain PUFAs is eicosapentaenoic acid (EPA). EPA is found in fungi and also in marine oils. Docosahexaenoic acid (DHA) is another important long-chain PUFA. DHA is most often found in fish oil and can also be purified from mammalian brain tissue. Arachidonic acid (ARA) is a third important long-chain PUFA. ARA is found in filamentous fungi and can also be purified from mammalian tissues including the liver and the adrenal glands.
ARA, EPA and/or DHA, for example, can be produced via either the alternate 4-8 desaturase/Δ9-elongase pathway or the conventional Δ6 pathway (see FIG. 1). Elongases, which are active on substrate fatty acids in the conventional 46 pathway for the production of long-chain PUFAs, particularly ARA, EPA and DHA, have previously been identified. The conventional Δ6 pathway for converting LA to DGLA and ALA to ω3-ETA utilizes the Δ6-desaturase enzyme to convert LA to GLA, and ALA to stearidonic acid (SDA), and the Δ6-elongase enzyme to convert GLA to DGLA, and SDA to ω3-ETA. However, in certain instances, the alternate Δ8-desaturase/Δ9-elongase pathway may be preferred over the conventional Δ6 pathway. For example, if particular residual omega-6 or omega-3 fatty acid intermediates, such as GLA or SDA, are not desired during production of DGLA, ω3-ETA, ARA, EPA, ω3-docosapentaenoic acid, ω6-docosapentaenoic acid, ADA and/or DHA, the alternate Δ8-desaturase/Δ9-elongase pathway may be used as an alternative to the conventional Δ6 pathway, to bypass GLA and SDA formation.
In the present disclosure, a new source of Δ9-elongase has been identified for the production of long-chain PUFAs, in particular DGLA, ETA, ARA, EPA, ω3-docosapentaenoic acid, ω6-docosapentaenoic acid, ADA and/or DHA. The Δ9-elongase enzyme of the present disclosure converts, for example, LA to ω6-EDA, and ALA to ω3-ETrA. The production of DGLA from ω6-EDA, and ARA from DGLA, is then catalyzed by a Δ8-desaturase and a Δ5-desaturase, respectively.